AbstractBackgroundAs major industrial poultry by‐products, chicken feet are considered as notable sources of several bioactive molecules. The current work covers the processing of chicken feet proteins as substrates to be hydrolysed by combinations of three commercial enzymes (Alcalase®, Flavourzyme® and Protana® Prime) during different hydrolysis periods and the evaluation of the identified peptides having antioxidant activity after simulated gastrointestinal digestion.ResultsEnzymatic hydrolysis with Alcalase® and Protana® Prime combination for 4 h resulted in the highest activities. Reversed‐phase high‐performance liquid chromatographic separation of the purified hydrolysate yielded three active fractions that were further identified by nano‐liquid chromatography–tandem mass spectrometry. The bioactivities of over 230 identified peptide sequences were estimated after simulated gastrointestinal digestion, and those peptides with the highest chance of exerting antioxidant activity were selected to be further synthesised and tested. In this sense, the synthesised dipeptides CF and GY showed the highest antioxidant capacity. CF presented IC50 values of 69.63 and 145.41 μmol L−1 in 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) assays, respectively. In contrast, GY IC50 values were 15.27 and 10.06 μmol L−1 in ABTS and ORAC assays, respectively. Significant differences (P < 0.05) were registered between peptides in the same antioxidant assays.ConclusionOverall, the findings emphasised the favourable impact of enzymatic hydrolysis with the obtaining of antioxidant peptides from poultry by‐products that could be evaluated as a safe and economical source to retard oxidation in food systems. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.